Synaptic loss and neuronal cell death correlate strongly with the degree of cognitive impairment in Alzheimer disease (Terry et al., Ann. Neurol. 30: 572-580 (1991); Hamos et al., Ann. Neurol. 39: 355 (1991); Dekosky & Scheff, Ann. Neurol. 27: 457-464 (1990)). These pathologic events are not yet defined at the biochemical level although many putative etiologic factors in Alzheimer disease share in common the potential for disrupting cellular calcium homeostasis (Khachaturian, Z. S., Aging 1(1): 17-34 (1989)), and some evidence exists for such disruption in Alzheimer tissues (Colvin, R. A., et al., Brain Res. 543: 139-147 (1991); Peterson, C., et al., N. Engl. J. Med. 312(16): 1063-1064 (1985); Peterson & Goldman, Proc. Natl. Acad. Sci. USA 83: 2758-2762 (1986); Rizopoulos, E., et al., Brain Res. Bull. 21: 825-828 (1988); Peterson, C., et al., Neurosci. Lett. 121: 239-243 (1991); Iacopino & Christakos, Proc. Natl. Acad. Sci. USA 87: 4078-4082 (1990); Gibson, G. E., et al., Biol. Psychiatry 22: 1079-1086 (1987)).
Calcium-activated neutral proteinases (CANP) are a family of proteases implicated in regulating aspects of signal transduction (Pontremoli, S., et al., Proc. Natl. Acad. Sci. USA 87: 3705-3707 (1990); Piggott, M. A., et al., Brain Res. 565: 42-47 (1991); Adunsky, A., et al., J. Neuroimmunology 33: 167-172 (1991); Suzuki & Ohno, Cell Struct. Funct. 15: 1-6 (1990); Murachi, T., Biochem. Internatl. 18(2): 263-294 (1989)). Limited proteolysis by CANPs is involved in regulating important enzymes, including calcium-dependent protein kinases and protein phosphatases and neurotransmitter enzymes, and in modifying the function of structural proteins of the membrane and membrane skeleton (Togari, A., et al., Biochem. Biophys. Res. Commun. 134(2): 749-754 (1968); Kishimoto, A., et al., J. Biol. Chem. 258: 1156-1164 (1983); Tallant, E. A., et al., Biochem. 27: 2205-2211 (1988); Nixon, R. A., Ann. N.Y. Acad. Sci. 568: 198-208 (1989)). Massive activation of CANPs, as occurs in excitatoxicity and ischemia, induces rapid irreversible neuronal injury (Siman, R., et al., J. Neurosci. 9: 1579-1590 (1989); Siman, R., in Neurotoxicity of Excitatory Amino Acids, A. Guidotti, ed., Raven Press, New York, pp. 145-161 (1990); Lee, K. S., et al., Proc. Natl. Acad. Sci. USA 88: 7233-7347 (1991)). However, previous reports indicate that total CANP activity is not significantly altered in Alzheimer brain (Kawashima, S., et al., Biomed. Res. 10(1): 17-23 (1989); Mantle & Perry, J. Neurol. Sci. 102: 220-224 (1991); Nilsson, E., et al., Neurobiol. Aging 11: 425-431 (1990)).
CANP exists in cells principally as an inactive precursor isoform (Suzuki & Ohno, Cell Struct. Funct. 15: 1-6 (1990); Suzuki, K., et al., FEBS Lett. 220: 271 (1987)), which is activated by autoproteolytic cleavage of an amino-terminal sequence in the presence of calcium (Suzuki, K., et al., J. Biochem. 90: 1787-1793 (1981); Inomata, M., et al., J. Biochem. 98: 407-416 (1985); Zimmerman & Schlaepfer, Biochim. Biophys. Acta 1078: 192-198 (1991); Suzuki, K., et al., FEBS Lett. 220: 271 (1987)). The action of CANP in vivo has been difficult to monitor by in vitro enzyme assay because the precursor form is also activated by the assay procedure, and activities are, in turn, influenced by instability of the enzyme as well as by various cytosolic inhibitory and activating factors (Mellgren & Murachi, eds., Intracellular Calcium-dependent Proteolysis, CRC Press, Boston, Mass., pp. 1-276 (1990)).